Abstract: The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a “phase diagram” in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA.

Abstract: Sweating responses were examined in five horses during a standardized exercise test (SET) in hot conditions (32-34 degrees C, 45-55% relative humidity) during 8 wk of exercise training (5 days/wk) in moderate conditions (19-21 degrees C, 45-55% relative humidity). SETs consisting of 7 km at 50% maximal O(2) consumption, determined 1 wk before training day (TD) 0, were completed on a treadmill set at a 6 degrees incline on TD0, 14, 28, 42, and 56. Mean maximal O(2) consumption, measured 2 days before each SET, increased 19% [TD0 to 42: 135 +/- 5 (SE) to 161 +/- 4 ml. kg(-1). min(-1)]. Peak sweating rate (SR) during exercise increased on TD14, 28, 42, and 56 compared with TD0, whereas SRs and sweat losses in recovery decreased by TD28. By TD56, end-exercise rectal and pulmonary artery temperature decreased by 0.9 +/- 0.1 and 1.2 +/- 0.1 degrees C, respectively, and mean change in body mass during the SET decreased by 23% (TD0: 10.1 +/- 0.9; TD56: 7.7 +/- 0.3 kg). Sweat Na(+) concentration during exercise decreased, whereas sweat K(+) concentration increased, and values for Cl(-) concentration in sweat were unchanged. Moderate-intensity training in cool conditions resulted in a 1.6-fold increase in sweating sensitivity evident by 4 wk and a 0.7 +/- 0.1 degrees C decrease in sweating threshold after 8 wk during exercise in hot, dry conditions. Altered sweating responses contributed to improved heat dissipation during exercise and a lower end-exercise core temperature. Despite higher SRs for a given core temperature during exercise, decreases in recovery SRs result in an overall reduction in sweat fluid losses but no change in total sweat ion losses after training.

Abstract: Equine thelaziosis caused by the eyeworm Thelazia lacrymalis is a parasitic disease transmitted by muscid flies. Although equine thelaziosis is known to have worldwide distribution, information on the epidemiology and presence of the intermediate hosts of T. lacrymalis is lacking. In the present work, a PCR-RFLP based assay on the first and/or second internal transcribed spacer (ITS1 and ITS2) of ribosomal DNA was developed for the detection of T. lacrymalis DNA in its putative vector(s). The sensitivity of the technique was also assessed. The restriction patterns obtained readily differentiated T. lacrymalis from four species of Musca (Diptera, Muscidae) (i.e. Musca autumnalis, Musca domestica, Musca larvipara and Musca osiris), which are potential vectors of equine eyeworms. The molecular assay presented herein is a useful tool to identify the intermediate host(s) of T. lacrymalis in natural conditions and to study its/their ecology and epidemiology.

Abstract: Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.

Abstract: To study nutrition and metabolism in the fetal pig, a chronic catheterization method was developed that allows blood sampling in arteries and veins, at both the umbilical and uterine sources, in the conscious, unstressed animal. A catheter was inserted in the fetal aorta through a femoral artery, and another one was introduced in the umbilical vein. A catheter was put in a femoral artery of the sow so that its end was in the abdominal aorta. A fourth catheter was placed in a uterine vein draining the fetoplacental unit studied. This procedure was applied to 18 Large White primiparous sows at 99 d of gestation. Blood samples were drawn simultaneously using the four catheters before a meal at 103 d of pregnancy, and glucose, insulin, lactate, and fructose were determinated. Glycemia was 2.5 times higher in the sow than in the fetus. The extraction coefficient of glucose by the fetus amounted to 14% of the umbilical supply. The insulin level in the fetal pig was very low ( < 5 microU/mL). Lactate and fructose seemed to originate from the placenta. Blood lactate was 2.6 times lower in the sow than in the fetus, and its extraction coefficient by the fetus amounted to 8%. Fructose in the fetal blood was 2.3 times higher than that of glucose. Fructose was not utilized by the pig fetus. The present results obtained in the fetal pig are comparable to the conclusions drawn from studies with other species.